Purification and properties of squirrel monkey (Saimiri sciureus) corticosteroid binding globulin.
Purification and properties of squirrel monkey (Saimiri sciureus) corticosteroid binding globulin.
复制标题
松鼠猴（Saimiri sciureus）皮质类固醇结合球蛋白的纯化和特性。
DOI：
10.1021/bi00407a046
复制
发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Siiteri,PK
中科院分区：
文献类型：
--
作者：
Kuhn,RW;VestWeber,C;Siiteri,PK
摘要：皮质类固醇结合球蛋白(CBG)是一种与糖皮质激素和孕激素高亲和力结合的血清糖蛋白，广泛分布于动物界。虽然CBG的电荷和大小特征在很大程度上在物种之间是保守的，但我们发现松鼠猴(Saimiri Scureus)血清中CBG在聚丙烯酰胺凝胶电泳或Sephadex层析分离过程中的行为与一个大约是大多数物种中发现的两倍大小的分子一致。为了更充分地了解这种差异的基础，我们通过顺序亲和和DEAE-Sepharose层析纯化了该蛋白。最终产物的得率大于60%，当在不同总丙烯酰胺浓度的聚丙烯酰胺凝胶中或在严重的蛋白质超载条件下，在pH 8.3的条件下进行电泳时，发现最终产物以单一均一条带的形式迁移。纯化的蛋白与起始血清中的蛋白具有相同的类固醇结合特异性。分离的CBG-甾体化合物的紫外吸收光谱表明，该蛋白质不含吡啶核苷酸辅因子或核酸。氨基酸分析表明，松鼠猴蛋白的组成与其他物种的CBG分子非常相似，但与白蛋白、血红蛋白或兔孕酮受体不同。与正常条件下观察到的单一蛋白质带不同，在十二烷基硫酸钠存在下的分离将纯蛋白质分解成两条带：一条在54000道尔顿，另一条在57000道尔顿。用可逆交联剂4-巯基丁基亚氨酸甲酯或二硫代二硫代丙酸二甲酯处理纯化材料后，在不加还原剂的条件下，凝胶上可检测到一条110000道尔顿的条带。该条带在电泳前用还原剂处理即可消除。这表明，与其他物种不同，松鼠猴CBG在其天然状态下以二聚体的形式存在。用放射免疫扩散和放射免疫分析技术对纯化的材料产生抗体，并检测其与其他物种血清的交叉反应。用放射免疫法检测时，仅观察到来自Titi猴的血清发生交叉反应。综上所述，我们的结果表明，新大陆猴CBG在大小和免疫学特征上都不同于其他物种。(皮质类固醇结合球蛋白(CBG)是一种血清糖蛋白，与天然糖皮质激素和孕酮高度结合。虽然人们普遍认为CBG的生理作用是调节血液中的游离皮质醇水平，因此它对靶细胞受体的可用性，但也有其他功能，包括激素的细胞内运输(Siiteri等人，1982)。这种蛋白质在生物学上的重要性从它在几乎每一个
Revised Manuscript Received November 19, 1987 abstract: Corticosteroid binding globulin (CBG), a serum glycoprotein which binds glucocorticoids and progestins with high affinity, is widely distributed throughout the animal world. Although its charge and size characteristics have largely been conserved across species, we found the behavior of CBG in squirrel monkey (Saimiri sciureus) serum during fractionation by polyacrylamide gel electrophoresis or Sephadex chromatography was consistent with a molecule about twice the size of that found in most species. To more fully understand the basis for thisdifference, we purified the protein by sequential affinity and DEAE-Sepharose chromatographies. The final product was obtained in greater than 60% yield and was found to migrate as a single homogeneous band when examined by electrophoresis at pH 8.3 in polyacrylamide gels varying total acrylamide concentration or under conditions of severe protein overload. The steroid binding specificity of the purified protein was identical with that of the protein in the starting serum. The ultraviolet absorption spectrum of the isolated CBG-steroid complexes revealed that the protein had no pyridine nucleotide cofactor or nucleic acid. Amino acid analyses showed that the composition of the squirrel monkey protein is quite similar to that of CBG molecules from other species but distinct from albumins, hemoglobin, or rabbit progesterone receptor. In contrast to the single protein band observed following electrophoresis under normal conditions, separations in the presence of sodium dodecyl sulfate (SDS) resolved the pure protein into two bands: one at 54000 daltons and one at 57 000 daltons. Following treatment of the purified material with the reversible cross-linking agents methyl 4-mercaptobutyrimidate or dimethyl dithiobis-(propionimidate), a band migrating at 110000 daltons was detected on SDS gels in the absence of reducing agents. This band was eliminated by treatment with reducingagents prior to electrophoresis. This shows that unlike other species, squirrel monkey CBG exists as a dimer in its native state. Antibodies were generated against the purified material and tested for cross-reactivity againstthe sera from other species by both radioimmunodiffusion and radioimmunoassay techniques. Only serum from titi monkeys was observed to cross-react when examined by radioimmunoassay. Taken together, our results suggest that New World monkey CBG’s are distinct from those of other species in both size and immunologic characteristics.(Corticosteroid binding globulin (CBG) is a serum glycoprotein that binds natural glucocorticoids and progesterone with high affinity. While it is generally believed that the physiologic role of CBG is regulation of free cortisol levels in blood and thus its availability to target cell receptors, other functions including intracellular transport of hormone have been suggested (Siiteri et al., 1982). The biologic importance of this protein is attested to by its presence in virtually every